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Abstract:

The present invention relates to modulators of cystic fibrosis
transmembrane conductance regulator ("CFTR"), compositions thereof, and
methods therewith. The present invention also relates to methods of
treating diseases using modulators of CFTR.

Claims:

1. A compound of Formula I ##STR00089## or pharmaceutically acceptable
salts thereof, wherein: ring A is selected from: ##STR00090## wherein:
R1 is --CH3, --CF3 or --CN; R2 is hydrogen,
--CH3, --CF3, --OH, or --CH2OH; R3 is hydrogen,
--CH3, --OCH3, or --CN; provided that both R2 and R3
are not simultaneously hydrogen; and one of X and Y is nitrogen and the
other is carbon.

2. The compound according to claim 1, wherein ring A is ##STR00091##

3. The compound according to claim 1, wherein ring A is ##STR00092##

4. The compound according to claim 1, wherein ring A is ##STR00093##

5. The compound according to claim 1, wherein ring A is ##STR00094##

6. The compound according to any one of claims 2-5, wherein R1 is
--CF.sub.3.

7. The compound according to any one of claims 2-5, wherein R1 is
--CN.

8. The compound according to any one of claims 2-5, wherein R1 is
--CH.sub.3.

9. The compound according to any one of claims 6-8, wherein R2 is
hydrogen.

10. The compound according to any one of claims 6-8, wherein R2 is
--CH.sub.3.

11. The compound according to any one of claims 6-8, wherein R2 is
--CF.sub.3.

12. The compound according to any one of claims 6-8, R2 is --OH.

13. The compound according to any one of claims 6-8, wherein R2 is
--CH2OH.

14. The compound according to any one of claims 9-13, wherein R3 is
hydrogen.

15. The compound according to any one of claims 9-13, wherein R3 is
--CH.sub.3.

16. The compound according to any one of claims 9-13, wherein R3 is
--OCH.sub.3.

17. The compound according to any one of claims 9-12, wherein R3 is
--CN.

18. The compound according to any one of claims 1-17 wherein X is
nitrogen.

19. The compound according to any one of claims 1-17 wherein Y is
nitrogen.

20. A compound selected from ##STR00095##

21. A pharmaceutical composition comprising a compound according to any
one of claims 1-20 and a pharmaceutically acceptable carrier or adjuvant.

22. The pharmaceutical composition according to claim 21, further
comprising an additional agent selected from a mucolytic agent, a
bronchodialator, an antibiotic, an anti-infective agent, an
anti-inflammatory agent, a CFTR modulator other than a compound of
formula (I), or a nutritional agent.

23. The pharmaceutical composition according to claim 22, wherein said
additional agent is a CFTR modulator other than a compound of formula
(I).

25. The method according to claim 24, wherein said disease is cystic
fibrosis.

26. A method of treating or lessening the severity of a disease in a
patient, wherein said disease is associated with reduced CFTR function
due to mutations in the gene encoding CFTR or environmental factors, said
method comprising the step of administering to said patient an effective
amount of a compound according to any one of claims 1-20.

28. A method of treating or lessening the severity of a disease in a
patient, wherein said disease is associated with normal CFTR function,
said method comprising the step of administering to said patient an
effective amount of a compound according to any one of claims 1-20.

31. A kit for use in measuring the activity of CFTR or a fragment thereof
in a biological sample in vitro or in vivo, comprising: (i) a composition
comprising a compound of Formula (I) according to claim 1; (ii)
instructions for: a) contacting the composition with the biological
sample; b) measuring activity of said CFTR or a fragment thereof.

32. The kit of claim 31, further comprising instructions for: a)
contacting an additional composition with the biological sample; b)
measuring the activity of said CFTR or a fragment thereof in the presence
of said additional compound, and c) comparing the activity of the CFTR in
the presence of the additional compound with the density of CFTR in the
presence of a composition of Formula (I).

33. A method of modulating CFTR activity in a biological sample
comprising the step of contacting said CFTR with a compound according to
any one of claims 1-20.

34. A process for preparing a compound of Formula (Ic): ##STR00096## or
pharmaceutically acceptable salts thereof, wherein the process comprises:
(a) reacting the acid of formula 1d with an amine of formula 2c to
provide a compound of formula (I) ##STR00097## wherein: ring A is
selected from: ##STR00098## wherein: R1 is --CH3, --CF3
or --CN; R2 is hydrogen, --CH3, --CF3, --OH, or
--CH2OH; R3 is hydrogen, --CH3, --OCH3, or --CN;
provided that both R2 and R3 are not simultaneously hydrogen,
and Ra is hydrogen or a silyl protecting group selected from the
group consisting of trimethylsilyl (TMS), tert-butyldiphenylsilyl
(TBDPS), tert-butyldimethylsilyl (TBDMS) triisopropylsilyl (TIPS), and
[2-(trimethylsilyl)ethoxy]methyl (SEM); and one of X and Y is nitrogen
and the other is carbon,

35. The process of claim 34, wherein the reaction of the acid of formula
1d with the amine of formula 2c occurs in a solvent in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and triethylamine or in a solvent in the
presence of propyl phosphonic acid cyclic anhydride (T3P®) and
pyridine.

39. The process of claim 34 further comprising a deprotection step to
remove the silyl protecting group when ring A is ##STR00099## wherein
Ra is a silyl protecting group, to generate a compound of formula I,
wherein ring A is ##STR00100##

40. The process of claim 34, wherein the amine of formula 2c is prepared
from a compound of formula 2a comprising the steps of: (a) reacting the
compound of formula 2a with an amine of formula 3 to provide the compound
of formula 2b ##STR00101## wherein: Hal is F, Cl, Br, or I; and the
amine of formula 3 is ##STR00102## and (b) reducing the compound of
formula 2b to the amine of formula 2c. ##STR00103##

41. The process of claim 40, wherein the amine of formula 3 in step (a)
is generated in situ from the amine hydrochloride salt.

42. The process of claim 40, wherein Ra is hydrogen or TBDMS.

43. The process of claim 42, wherein Ra is TBDMS.

44. The process of claim 40, wherein step (a) occurs in a polar aprotic
solvent in the presence of a tertiary amine base.

45. The process of claim 44, wherein step (a) occurs in acetonitrile in
the presence of triethylamine.

46. The process of claim 40, wherein the reaction temperature of step (a)
is between approximately 75.degree. C. and approximately 85.degree. C.

47. The process of claim 40, wherein the reaction time is between
approximately 2 and approximately 30 hours.

48. The process of claim 40, wherein step (b) occurs in a polar protic
solvent in the presence of a palladium catalyst.

49. The process of claim 48, wherein the solvent in step (b) comprises
methanol or ethanol.

50. The process of claim 40, wherein step (b) occurs in a polar protic
solvent in the presence of Fe and FeSO4 or Zn and AcOH.

51. The process of claim 50, wherein the polar protic solvent is water.

52. A process for preparing a compound of formula Ic, ##STR00104## or
pharmaceutically acceptable salts thereof, comprising the steps of: (a)
reacting a compound of formula 2a with an amine of formula 3 to provide a
compound of formula 2b ##STR00105## (b) converting the compound of
formula 2b to the amine of formula 2c via hydrogenation ##STR00106##
and (c) reacting the amine of formula 2c with an acid of formula 1d to
provide a compound of formula Ic ##STR00107## wherein Hal is F, Cl, Br,
or I; the amine of formula 3 is ##STR00108## and ring A is selected
from: ##STR00109## wherein R1 is --CH3, --CF3 or --CN;
R2 is hydrogen, --CH3, --CF3, --OH, or --CH2OH;
R3 is hydrogen, --CH3, --OCH3, or --CN; provided that both
R2 and R3 are not simultaneously hydrogen; Ra is hydrogen
or a silyl protecting group selected from the group consisting of
trimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS),
tert-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), and
[2-(trimethylsilyl)ethoxy]methyl (SEM); and one of X and Y is nitrogen
and the other is carbon.

53. The process of claim 52, wherein the amine of formula 3 ##STR00110##
in step (a) is generated in situ from the amine hydrochloride salt.

54. The process of claim 53, wherein Ra is hydrogen or TBDMS.

55. The process of claim 54, wherein Ra is TBDMS.

56. The process of claim 52, wherein step (a) occurs in a polar aprotic
solvent in the presence of a tertiary amine base.

57. The process of claim 56, wherein step (a) occurs in acetonitrile in
the presence of triethylamine.

58. The process of claim 52, wherein the reaction temperature of step (a)
is between approximately 75.degree. C. and approximately 85.degree. C.

59. The process of claim 52, wherein the reaction time is between
approximately 2 and approximately 30 hours.

60. The process of claim 52, wherein step (b) occurs in a polar protic
solvent in the presence of a palladium catalyst.

61. The process of claim 60, wherein the solvent in step (b) comprises
methanol or ethanol.

62. The process of claim 52, wherein step (b) occurs in a polar protic
solvent in the presence of Fe and FeSO4 or Zn and AcOH.

63. The process of claim 62, wherein the polar protic solvent is water.

64. The process of claim 52, wherein step (c) occurs in a solvent in the
presence of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and triethylamine or in a solvent in the
presence of propyl phosphonic acid cyclic anhydride (T3P®) and
pyridine.

68. The process of claim 52 further comprising a deprotection reaction
when ring A is ##STR00111## wherein Ra is a silyl protecting
group, to generate a compound of formula I wherein ring A is
##STR00112##

69. A compound which is ##STR00113## wherein ring A is selected from:
##STR00114## wherein: R1 is --CH3, --CF3 or --CN; Ra
is hydrogen or a silyl protecting group selected from the group
consisting of trimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS),
tert-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), and
[2-(trimethylsilyl)ethoxy]methyl (SEM); and one of X and Y is nitrogen
and the other is carbon.

70. A compound which is ##STR00115## wherein ring A is selected from:
##STR00116## wherein: R1 is --CH3, --CF3 or --CN; Ra
is hydrogen or a silyl protecting group selected from the group
consisting of trimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS),
tert-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), and
[2-(trimethylsilyl)ethoxy]methyl (SEM); and one of X and Y is nitrogen
and the other is carbon.

71. A compound of formula Ic ##STR00117## or pharmaceutically
acceptable salts thereof, wherein: ring A is selected from ##STR00118##
wherein R1 is --CH3, --CF3 or --CN; R2 is hydrogen,
--CH3, --CF3, --OH, or --CH2OH; R3 is hydrogen,
--CH3, --OCH3, or --CN; provided that both R2 and R3
are not simultaneously hydrogen, and Ra is a silyl protecting group
selected from the group consisting of trimethylsilyl (TMS),
tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBDMS),
triisopropylsilyl (TIPS), and [2-(trimethylsilyl)ethoxy]methyl (SEM); and
one of X and Y is nitrogen and the other is carbon.

72. A compound of formula I ##STR00119## or pharmaceutically acceptable
salts thereof, wherein: ring A is selected from: ##STR00120## wherein:
R1 is --CH3, --CF3 or --CN; R2 is hydrogen,
--CH3, --CF3, --OH, or --CH2OH; R3 is hydrogen,
--CH3, --OCH3, or --CN; provided that both R2 and R3
are not simultaneously hydrogen, and one of X and Y is nitrogen and the
other is carbon, made by the process of any of claims 52-68.

73. A compound which is selected from the group consisting of
##STR00121## made by the process of any of claims 52-68.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority under 35
U.S.C. §119 to U.S. Provisional Application Ser. No. 61/107,844,
filed Oct. 23, 2008 and entitled "MODULATORS OF CYSTIC FIBROSIS
TRANSMEMBRANE CONDUCTANCE REGULATOR," the entire contents of which is
incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to modulators of cystic fibrosis
transmembrane conductance regulator ("CFTR"), compositions thereof, and
methods therewith. The present invention also relates to methods of
treating diseases using modulators of CFTR.

BACKGROUND OF THE INVENTION

[0003] ATP cassette transporters are a family of membrane transporter
proteins that regulate the transport of a wide variety of pharmacological
agents, potentially toxic drugs, and xenobiotics, as well as anions. They
are homologous membrane proteins that bind and use cellular adenosine
triphosphate (ATP) for their specific activities. Some of these
transporters were discovered as multidrug resistance proteins (like the
MDR1-P glycoprotein, or the multidrug resistance protein, MRP1),
defending malignant cancer cells against chemotherapeutic agents. To
date, 48 such transporters have been identified and grouped into 7
families based on their sequence identity and function.

[0004] One member of the ATP cassette transporters family commonly
associated with disease is the cAMP/ATP-mediated anion channel, CFTR.
CFTR is expressed in a variety of cells types, including absorptive and
secretory epithelia cells, where it regulates anion flux across the
membrane, as well as the activity of other ion channels and proteins. In
epithelial cells, normal functioning of CFTR is critical for the
maintenance of electrolyte transport throughout the body, including
respiratory and digestive tissue. CFTR is composed of approximately 1480
amino acids that encode a protein made up of a tandem repeat of
transmembrane domains, each containing six transmembrane helices and a
nucleotide binding domain. The two transmembrane domains are linked by a
large, polar, regulatory (R)-domain with multiple phosphorylation sites
that regulate channel activity and cellular trafficking.

[0005] The gene encoding CFTR has been identified and sequenced (See
Gregory, R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al.
(1990) Nature 347:358-362), Riordan, J. R. et al. (1989) Science
245:1066-1073). A defect in this gene causes mutations in CFTR resulting
in cystic fibrosis ("CF"), the most common fatal genetic disease in
humans. Cystic fibrosis affects approximately one in every 2,500 infants
in the United States. Within the general United States population, up to
10 million people carry a single copy of the defective gene without
apparent ill effects. In contrast, individuals with two copies of the CF
associated gene suffer from the debilitating and fatal effects of CF,
including chronic lung disease.

[0006] In patients with cystic fibrosis, mutations in CFTR endogenously
expressed in respiratory epithelia lead to reduced apical anion secretion
causing an imbalance in ion and fluid transport. The resulting decrease
in anion transport contributes to enhanced mucus accumulation in the lung
and the accompanying microbial infections that ultimately cause death in
CF patients. In addition to respiratory disease, CF patients typically
suffer from gastrointestinal problems and pancreatic insufficiency that,
if left untreated, results in death. In addition, the majority of males
with cystic fibrosis are infertile and fertility is decreased among
females with cystic fibrosis. In contrast to the severe effects of two
copies of the CF associated gene, individuals with a single copy of the
CF associated gene exhibit increased resistance to cholera and to
dehydration resulting from diarrhea--perhaps explaining the relatively
high frequency of the CF gene within the population.

[0007] Sequence analysis of the CFTR gene of CF chromosomes has revealed a
variety of disease causing mutations (Cutting, G. R. et al. (1990) Nature
346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem, B-S. et
al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc. Natl.
Acad. Sci. USA 87:8447-8451). To date, more than 1000 disease causing
mutations in the CF gene have been identified
(http://www.genet.sickkids.on.calcftr/). The most prevalent mutation is a
deletion of phenylalanine at position 508 of the CFTR amino acid
sequence, and is commonly referred to as ΔF508-CFTR. This mutation
occurs in approximately 70 percent of the cases of cystic fibrosis and is
associated with a severe disease.

[0008] The deletion of residue 508 in ΔF508-CFTR prevents the
nascent protein from folding correctly. This results in the inability of
the mutant protein to exit the ER, and traffic to the plasma membrane. As
a result, the number of channels present in the membrane is far less than
observed in cells expressing wild-type CFTR. In addition to impaired
trafficking, the mutation results in defective channel gating. Together,
the reduced number of channels in the membrane and the defective gating
lead to reduced anion transport across epithelia, leading to defective
ion and fluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727).
Studies have shown, however, that the reduced numbers of ΔF508-CFTR
in the membrane are functional, albeit less than wild-type CFTR. (Dolmans
et al. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk
and Foskett (1995), J. Cell. Biochem. 270: 12347-50). In addition to
ΔF508-CFTR, R117H-CFTR and G551D-CFTR, other disease causing
mutations in CFTR that result in defective trafficking, synthesis, and/or
channel gating, could be up- or down-regulated to alter anion secretion
and modify disease progression and/or severity.

[0009] Although CFTR transports a variety of molecules in addition to
anions, it is clear that this role (the transport of anions, chloride and
bicarbonate) represents one element in an important mechanism of
transporting ions and water across the epithelium. The other elements
include the epithelial Na+ channel, ENaC, Na+/2Cl.sup.-/K+
co-transporter, Na+--K+-ATPase pump and the basolateral
membrane K+ channels, that are responsible for the uptake of
chloride into the cell.

[0010] These elements work together to achieve directional transport
across the epithelium via their selective expression and localization
within the cell. Chloride absorption takes place by the coordinated
activity of ENaC and CFTR present on the apical membrane and the
Na+--K+-ATPase pump and Cl channels expressed on the
basolateral surface of the cell. Secondary active transport of chloride
from the luminal side leads to the accumulation of intracellular
chloride, which can then passively leave the cell via Cl.sup.- ion
channels, resulting in a vectorial transport. Arrangement of
Na+/2Cl.sup.-/K+ co-transporter, Na+--K+-ATPase pump
and the basolateral membrane K+ channels on the basolateral surface
and CFTR on the luminal side coordinate the secretion of chloride via
CFTR on the luminal side. Because water is probably never actively
transported itself, its flow across epithelia depends on tiny
transepithelial osmotic gradients generated by the bulk flow of sodium
and chloride.

[0012] Mutations in CFTR that are associated with moderate CFTR
dysfunction are also evident in patients with conditions that share
certain disease manifestations with CF but do not meet the diagnostic
criteria for CF. These include congenital bilateral absence of the vas
deferens, idiopathic chronic pancreatitis, chronic bronchitis, and
chronic rhinosinusitis. Other diseases in which mutant CFTR is believed
to be a risk factor along with modifier genes or environmental factors
include primary sclerosing cholangitis, allergic bronchopulmonary
aspergillosis, and asthma.

[0013] Cigarette smoke, hypoxia, and environmental factors that induce
hypoxic signaling have also been demonstrated to impair CFTR function and
may contribute to certain forms of respiratory disease, such as chronic
bronchitis. Diseases that may be due to defective CFTR function but do
not meet the diagnostic criteria for CF are characterized as CFTR-related
diseases.

[0014] In addition to cystic fibrosis, modulation of CFTR activity may be
beneficial for other diseases not directly caused by mutations in CFTR,
such as secretory diseases and other protein folding diseases mediated by
CFTR. CFTR regulates chloride and bicarbonate flux across the epithelia
of many cells to control fluid movement, protein solubilization, mucus
viscosity, and enzyme activity. Defects in CFTR can cause blockage of the
airway or ducts in many organs, including the liver and pancreas.
Potentiators are compounds that enhance the gating activity of CFTR
present in the cell membrane. Any disease which involves thickening of
the mucus, impaired fluid regulation, impaired mucus clearance, or
blocked ducts leading to inflammation and tissue destruction could be a
candidate for potentiators.

[0015] These include, but are not limited to, chronic obstructive
pulmonary disease (COPD), asthma, smoke induced COPD, chronic bronchitis,
rhinosinusitis, constipation, dry eye disease, and Sjogren's Syndrome,
gastro-esophageal reflux disease, gallstones, rectal prolapse, and
inflammatory bowel disease. COPD is characterized by airflow limitation
that is progressive and not fully reversible. The airflow limitation is
due to mucus hypersecretion, emphysema, and bronchiolitis. Activators of
mutant or wild-type CFTR offer a potential treatment of mucus
hypersecretion and impaired mucociliary clearance that is common in COPD.
Specifically, increasing anion secretion across CFTR may facilitate fluid
transport into the airway surface liquid to hydrate the mucus and
optimized periciliary fluid viscosity. This would lead to enhanced
mucociliary clearance and a reduction in the symptoms associated with
COPD. In addition, by preventing ongoing infection and inflammation due
to improved airway clearance, CFTR modulators may prevent or slow the
parenchimal destruction of the airway that characterizes emphysema and
reduce or reverse the increase in mucus secreting cell number and size
that underlyses mucus hypersecretion in airway diseases. Dry eye disease
is characterized by a decrease in tear aqueous production and abnormal
tear film lipid, protein and mucin profiles. There are many causes of dry
eye, some of which include age, Lasik eye surgery, arthritis,
medications, chemical/thermal burns, allergies, and diseases, such as
cystic fibrosis and Sjogren's syndrome. Increasing anion secretion via
CFTR would enhance fluid transport from the corneal endothelial cells and
secretory glands surrounding the eye to increase corneal hydration. This
would help to alleviate the symptoms associated with dry eye disease.
Sjogrens's syndrome is an autoimmune disease in which the immune system
attacks moisture-producing glands throughout the body, including the eye,
mouth, skin, respiratory tissue, liver, vagina, and gut. Symptoms,
include, dry eye, mouth, and vagina, as well as lung disease. The disease
is also associated with rheumatoid arthritis, systemic lupus, systemic
sclerosis, and polymypositis/dermatomyositis. Defective protein
trafficking is believed to cause the disease, for which treatment options
are limited. Modulators of CFTR activity may hydrate the various organs
afflicted by the disease and may help to alleviate the associated
symptoms. Individuals with cystic fibrosis have recurrent episodes of
intestinal obstruction and higher incidences of rectal polapse,
gallstones, gastro-esophageal reflux disease, GI malignancies, and
inflammatory bowel disease, indicating that CFTR function may play an
important role in preventing such diseases.

[0018] In addition to up-regulation of CFTR activity, reducing anion
secretion by CFTR modulators may be beneficial for the treatment of
secretory diarrheas, in which epithelial water transport is dramatically
increased as a result of secretagogue activated chloride transport. The
mechanism involves elevation of cAMP and stimulation of CFTR.

[0019] Although there are numerous causes of diarrhea, the major
consequences of diarrheal diseases, resulting from excessive chloride
transport are common to all, and include dehydration, acidosis, impaired
growth and death. Acute and chronic diarrheas represent a major medical
problem in many areas of the world. Diarrhea is both a significant factor
in malnutrition and the leading cause of death (5,000,000 deaths/year) in
children less than five years old.

[0020] Secretory diarrheas are also a dangerous condition in patients with
acquired immunodeficiency syndrome (AIDS) and chronic inflammatory bowel
disease (IBD). Sixteen million travelers to developing countries from
industrialized nations every year develop diarrhea, with the severity and
number of cases of diarrhea varying depending on the country and area of
travel.

[0021] Accordingly, there is a need for potent and selective CFTR
potentiators of wild-type and mutant forms of human CFTR. These mutant
CFTR forms include, but are not limited to, ΔF508del, G551D, R117H,
2789+5G->A.

[0022] There is also a need for modulators of CFTR activity, and
compositions thereof, which can be used to modulate the activity of the
CFTR in the cell membrane of a mammal.

[0023] There is a need for methods of treating diseases caused by mutation
in CFTR using such modulators of CFTR activity.

[0024] There is a need for methods of modulating CFTR activity in an ex
vivo cell membrane of a mammal.

SUMMARY OF THE INVENTION

[0025] It has now been found that compounds of this invention, and
pharmaceutically acceptable compositions thereof, are useful as
modulators of CFTR activity. These compounds have the general Formula
(I):

##STR00001##

[0026] or pharmaceutically acceptable salts thereof, wherein R1,
R2, R3, X, Y, and A are described generally and in classes and
subclasses below.

[0027] These compounds and pharmaceutically acceptable compositions are
useful for treating or lessening the severity of a variety of diseases,
disorders, or conditions associated with mutations in CFTR.

DETAILED DESCRIPTION OF THE INVENTION

General Description of Compounds of the Invention

[0028] The present invention relates to compounds of Formula (I) useful as
modulators of CFTR activity:

[0031] wherein: [0032] R1 is --CH3, --CF3 or --CN; [0033]
R2 is hydrogen, --CH3, --CF3, --OH, or --CH2OH;
[0034] R3 is hydrogen, --CH3, --OCH3, or --CN; [0035]
provided that, [0036] both R2 and R3 are not simultaneously
hydrogen; [0037] X is carbon or nitrogen; [0038] Y is carbon or
nitrogen; [0039] provided that [0040] both X and Y are not
simultaneously nitrogen.

COMPOUNDS AND DEFINITIONS

[0041] Compounds of this invention include those described generally
above, and are further illustrated by the classes, subclasses, and
species disclosed herein. As used herein, the following definitions shall
apply unless otherwise indicated.

[0042] The term "ABC-transporter" as used herein means an ABC-transporter
protein or a fragment thereof comprising at least one binding domain,
wherein said protein or fragment thereof is present in vivo or in vitro.
The term "binding domain" as used herein means a domain on the
ABC-transporter that can bind to a modulator. See, e.g., Hwang, T. C. et
al., J. Gen. Physiol. (1998): 111(3), 477-90.

[0044] The term "modulating" as used herein means increasing or decreasing
by a measurable amount.

[0045] The term "normal CFTR" or "normal CFTR function" as used herein
means wild-type like CFTR without any impairment due to environmental
factors such as smoking, pollution, or anything that produces
inflammation in the lungs.

[0046] The term "reduced CFTR" or "reduced CFTR function" as used herein
means less than normal CFTR or less than normal CFTR function. For
purposes of this invention, the chemical elements are identified in
accordance with the Periodic Table of the Elements, CAS version, Handbook
of Chemistry and Physics, 75th Ed. Additionally, general principles
of organic chemistry are described in "Organic Chemistry", Thomas
Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced
Organic Chemistry", 5th Ed., Ed.: Smith, M. B. and March, J., John
Wiley & Sons, New York: 2001, the entire contents of which are hereby
incorporated by reference.

[0047] Combinations of substituents envisioned by this invention are
preferably those that result in the formation of stable or chemically
feasible compounds. The term "stable", as used herein, refers to
compounds that are not substantially altered when subjected to conditions
to allow for their production, detection, and preferably their recovery,
purification, and use for one or more of the purposes disclosed herein.
In some embodiments, a stable compound or chemically feasible compound is
one that is not substantially altered when kept at a temperature of
40° C. or less, in the absence of moisture or other chemically
reactive conditions, for at least a week.

[0048] The term "protecting group", as used herein, refers to an agent
used to temporarily to block one or more desired reactive sites in a
multifunctional compound. In certain embodiments, a protecting group has
one or more, or preferably all, of the following characteristics: a)
reacts selectively in good yield to give a protected substrate that is
stable to the reactions occurring at one or more of the other reactive
sites; and b) is selectively removable in good yield by reagents that do
not attack the regenerated functional group. Exemplary protecting groups
are detailed in Greene, T. W., Wuts, P. G in "Protective Groups in
Organic Synthesis", Third Edition, John Wiley & Sons, New York: 1999, and
other editions of this book, the entire contents of which are hereby
incorporated by reference.

[0049] Unless otherwise stated, structures depicted herein are also meant
to include all isomeric (e.g., enantiomeric, diastereomeric, and
geometric (or conformational)) forms of the structure; for example, the R
and S configurations for each asymmetric center, (Z) and (E) double bond
isomers, and (Z) and (E) conformational isomers. Therefore, single
stereochemical isomers as well as enantiomeric, diastereomeric, and
geometric (or conformational) mixtures of the present compounds are
within the scope of the invention. Unless otherwise stated, all
tautomeric forms of the compounds of the invention are within the scope
of the invention; e.g., compounds of Formula (I) may exist as tautomers:

##STR00004##

[0050] Additionally, unless otherwise stated, structures depicted herein
are also meant to include compounds that differ only in the presence of
one or more isotopically enriched atoms. For example, compounds having
the present structures except for the replacement of hydrogen by
deuterium or tritium, or the replacement of a carbon by a 13C- or
14C-enriched carbon are within the scope of this invention. Such
compounds are useful, for example, as analytical tools or probes in
biological assays.

Description of Exemplary Compounds:

[0051] According to one embodiment, the present invention provides
compounds of Formula (I):

[0053] ##STR00006## [0054] wherein: [0055] R1 is --CH3,
--CF3 or --CN; [0056] R2 is hydrogen, --CH3, --CF3,
--OH, or --CH2OH; [0057] R3 is hydrogen, --CH3,
--OCH3, or --CN; [0058] provided that, [0059] both R2 and
R3 are not simultaneously hydrogen; and [0060] one of X and Y is
nitrogen and the other is carbon.

[0061] In one aspect of this embodiment, the compound is the free amine.

[0062] In another aspect, the compound is in a pharmaceutically acceptable
salt., such as the HCl salt.

[0063] In one embodiment, ring A is

##STR00007##

[0064] In one embodiment, ring A is

##STR00008##

[0065] In another embodiment, ring A is

##STR00009##

[0066] In yet another embodiment, ring A is

##STR00010##

[0067] In one embodiment, R1 is --CH3.

[0068] In one embodiment, R1 is --CF3.

[0069] In another embodiment, R1 is --CN.

[0070] In one embodiment, R2 is --CH3.

[0071] In another embodiment, R2 is --CF3.

[0072] In another embodiment, R2 is --OH.

[0073] In another embodiment, R2 is --CH2OH.

[0074] In one embodiment, R3 is --CH3.

[0075] In one embodiment, R3 is --OCH3.

[0076] In another embodiment, R3 is --CN.

[0077] In one embodiment, R2 is hydrogen; and R3 is --CH3,
--OCH3, or --CN.

[0078] In several embodiments of Formula (I), Ring A is

##STR00011##

R1 is --CF3, R2 is hydrogen; and R3 is --CH3,
--OCH3, or --CN. In other embodiments, R1 is --CH3. In
other embodiments, R1 is --CN. In one embodiment, R3 is
--OCH3. Or, R3 is --CH3. Or, R3 is --CN.

[0079] In further embodiments of Formula (I), Ring A is

##STR00012##

R1 is --CF3, R2 is --CH3, --CF3, --OH, or
--CH2OH, and R3 is hydrogen. In other embodiments, R1 is
--CH3. In other embodiments, R1 is --CN. In one embodiment,
R2 is --CH3. Or, R2 is --CF3. Or, R2 is --OH.
Or, R2 is --CH2OH.

[0080] In another embodiment, R2 is --CH3, --CF3, --OH, or
--CH2OH; and R3 is hydrogen.

[0081] In several embodiments of Formula (I), Ring A is

##STR00013##

R1 is --CF3, R2 is hydrogen; and R3 is --CH3,
--OCH3, or --CN. In other embodiments, R1 is --CH3. In
other embodiments, R1 is --CN. In one embodiment, R3 is
--CH3. Or, R3 is --OCH3. Or, R3 is --CN.

[0082] In several embodiments of Formula (I), Ring A is

##STR00014##

R1 is --CF3, R2 is hydrogen; and R3 is --CH3,
--OCH3, or --CN. In other embodiments, R1 is --CH3. In
other embodiments, R1 is --CN. In one embodiment, R3 is
--CH3. Or, R3 is --OCH3. Or, R3 is --CN.

[0083] In further embodiments of Formula (I), Ring A is

##STR00015##

R1 is --CF3, R2 is --CH3, --CF3, --OH, or
--CH2OH, and R3 is hydrogen. In other embodiments, R1 is
--CH3. In other embodiments, R1 is --CN. In one embodiment,
R2 is --CH3. Or, R2 is --CF3. Or, R2 is --OH.
Or, R2 is --CH2OH.

[0084] In several embodiments of Formula (I), Ring A is

##STR00016##

R1 is --CF3, R2 is hydrogen; and R3 is --CH3,
--OCH3, or --CN. In other embodiments, R1 is --CH3. In
other embodiments, R1 is --CN. In one embodiment, R3 is
--CH3. Or, R3 is --OCH3. Or, R3 is --CN.

[0085] In further embodiments of Formula (I), Ring A is

##STR00017##

R1 is --CF3, R2 is --CH3, --CF3, --OH, or
--CH2OH, and R3 is hydrogen. In other embodiments, R1 is
--CH3. In other embodiments, R1 is --CN. In one embodiment,
R2 is --CH3. Or, R2 is --CF3. Or, R2 is --OH.
Or, R2 is --CH2OH.

[0086] In several embodiments of Formula (I), Ring A is

##STR00018##

R1 is --CF3, R2 is hydrogen; and R3 is --CH3,
--OCH3, or --CN. In other embodiments, R1 is --CH3. In
other embodiments, R1 is --CN. In one embodiment, R3 is
--CH3. Or, R3 is --OCH3. Or, R3 is --CN.

[0087] In further embodiments of Formula (I), Ring A is

##STR00019##

R1 is --CF3, R2 is --CH3, --CF3, --OH, or
--CH2OH, and R3 is hydrogen. In other embodiments, R1 is
--CH3. In other embodiments, R1 is --CN. In one embodiment,
R2 is --CH3. Or, R2 is --CF3. Or, R2 is --OH.
Or, R2 is --CH2OH.

[0088] According to another embodiment, the present invention provides
compounds of Formula (Ia):

[0111] In one embodiment of Formula (Ia), R2 is hydrogen; and R3
is --CH3, --OCH3, or --CN.

[0112] In another embodiment of Formula (Ia), R2 is --CH3,
--CF3, --OH, or --CH2OH; and R3 is hydrogen.

[0113] In several embodiments of Formula (Ia), Ring A is

##STR00026##

R1 is --CF3, R2 is hydrogen; and R3 is --CH3,
--OCH3, or --CN. In other embodiments, R1 is --CH3. In
other embodiments, R1 is --CN. In one embodiment, R3 is
--OCH3. Or, R3 is --CH3. Or, R3 is --CN.

[0114] In further embodiments of Formula (Ia), Ring A is

##STR00027##

R1 is --CF3, R2 is --CH3, --CF3, --OH, or
--CH2OH, and R3 is hydrogen. In other embodiments, R1 is
--CH3. In other embodiments, R1 is --CN. In one embodiment,
R2 is --CH3. Or, R2 is --CF3. Or, R2 is --OH.
Or, R2 is --CH2OH.

[0115] In several embodiments of Formula (Ia), Ring A is

##STR00028##

R1 is --CF3, R2 is hydrogen; and R3 is --CH3,
--OCH3, or --CN. In other embodiments, R1 is --CH3. In
other embodiments, R1 is --CN. In one embodiment, R3 is
--CH3. Or, R3 is --OCH3. Or, R3 is --CN.

[0116] In further embodiments of Formula (Ia), Ring A is

##STR00029##

R1 is --CF3, R2 is --CH3, --CF3, --OH, or
--CH2OH, and R3 is hydrogen. In other embodiments, R1 is
--CH3. In other embodiments, R1 is --CN. In one embodiment,
R2 is --CH3. Or, R2 is --CF3. Or, R2 is --OH.
Or, R2 is --CH2OH.

[0117] In several embodiments of Formula (Ia), Ring A is

##STR00030##

R1 is --CF3, R2 is hydrogen; and R3 is --CH3,
--OCH3, or --CN. In other embodiments, R1 is --CH3. In
other embodiments, R1 is --CN. In one embodiment, R3 is
--CH3. Or, R3 is --OCH3. Or, R3 is --CN.

[0118] In further embodiments of Formula (Ia), Ring A is

##STR00031##

R1 is --CF3, R2 is --CH3, --CF3, --OH, or
--CH2OH, and R3 is hydrogen. In other embodiments, R1 is
--CH3. In other embodiments, R1 is --CN. In one embodiment,
R2 is --CH3. Or, R2 is --CF3. Or, R2 is --OH.
Or, R2 is --CH2OH.

[0119] In several embodiments of Formula (Ia), Ring A is

##STR00032##

R is --CF3, R2 is hydrogen; and R3 is --CH3,
--OCH3, or --CN. In other embodiments, R1 is --CH3. In
other embodiments, R1 is --CN. In one embodiment, R3 is
--CH3. Or, R3 is --OCH3. Or, R3 is --CN.

[0120] In further embodiments of Formula (Ia), Ring A is

##STR00033##

R1 is --CF3, R2 is --CH3, --CF3, --OH, or
--CH2OH, and R3 is hydrogen. In other embodiments, R1 is
--CH3. In other embodiments, R1 is --CN. In one embodiment,
R2 is --CH3. Or, R2 is --CF3. Or, R2 is --OH.
Or, R2 is --CH2OH.

[0121] According to another embodiment, the present invention provides
compounds of Formula (Ib):

[0144] In one embodiment of Formula (Ib), R2 is hydrogen; and R3
is --CH3, --OCH3, or --CN.

[0145] In another embodiment of Formula (Ib), R2 is --CH3,
--CF3, --OH, or --CH2OH; and R3 is hydrogen.

[0146] In several embodiments of Formula (Ib), ring A is

##STR00040##

R1 is --CF3, R2 is hydrogen; and R3 is --CH3,
--OCH3, - or --CN. In other embodiments, R1 is --CN. In other
embodiments, R1 is --CH3. In one embodiment, R3 is
--OCH3. Or, R3 is --CH3. Or, R3 is --CN.

[0147] In further embodiments of Formula (Ib), ring A is

##STR00041##

R1 is --CF3, R2 is --CH3, --CF3, --OH, or
--CH2OH, and R3 is hydrogen. In other embodiments, R1 is
--CN. In other embodiments, R1 is --CH3. In one embodiment,
R2 is --CH3. Or, R2 is --CF3. Or, R2 is --OH.
Or, R2 is --CH2OH.

[0148] In several embodiments of Formula (Ib), ring A is

##STR00042##

R1 is --CF3, R2 is hydrogen; and R3 is --CH3,
--OCH3, or --CN. In other embodiments, R1 is --CN. In other
embodiments, R1 is --CH3. In one embodiment, R3 is
--CH3. Or, R3 is --OCH3. Or, R3 is --CN.

[0149] In further embodiments of Formula (Ib), ring A is

##STR00043##

R1 is --CF3, R2 is --CH3, --CF3, --OH, or
--CH2OH, and R3 is hydrogen. In other embodiments, R1 is
--CN. In other embodiments, R1 is --CH3. In one embodiment,
R2 is --CH3. Or, R2 is --CF3. Or, R2 is --OH.
Or, R2 is --CH2OH.

[0150] In several embodiments of Formula (Ib) ring A is

##STR00044##

R1 is --CF3, R2 is hydrogen; and R3 is --CH3,
--OCH3, or --CN. In other embodiments, R1 is --CN. In other
embodiments, R1 is --CH3. In one embodiment, R3 is
--CH3. Or, R3 is --OCH3. Or, R3 is --CN.

[0151] In further embodiments of Formula (Ib), ring A is

##STR00045##

R1 is --CF3, R2 is --CH3, --CF3, --OH, or
--CH2OH, and R3 is hydrogen. In other embodiments, R1 is
--CN. In other embodiments, R1 is --CH3. In one embodiment,
R2 is --CH3. Or, R2 is --CF3. Or, R2 is --OH.
Or, R2 is --CH2OH.

[0152] In several embodiments of Formula (Ib), ring A is

##STR00046##

R1 is --CF3, R2 is hydrogen; and R3 is --CH3,
--OCH3, or --CN. In other embodiments, R1 is --CN. In other
embodiments, R1 is --CH3. In one embodiment, R3 is
--CH3. Or, R3 is --OCH3. Or, R3 is --CN.

[0153] In further embodiments of Formula (Ib), ring A is

##STR00047##

R1 is --CF3, R2 is --CH3, --CF3, --OH, or
--CH2OH, and R3 is hydrogen. In other embodiments, R1 is
--CN. In other embodiments, R1 is --CH3. In one embodiment,
R2 is --CH3. Or, R2 is --CF3. Or, R2 is --OH.
Or, R2 is --CH2OH.

[0154] Representative compounds of the present invention are set forth
below in Table 1 below.

TABLE-US-00001
TABLE 1
##STR00048## 1
##STR00049## 2
##STR00050## 3

General Synthetic Schemes

[0155] Compounds of the present invention are readily prepared by methods
known in the art. Illustrated in Schemes in the Examples herein below are
exemplary methods for the preparation of compounds of the present
invention.

Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

[0156] In one aspect of the present invention, pharmaceutically acceptable
compositions are provided, wherein these compositions comprise any of the
compounds as described herein, and optionally comprise a pharmaceutically
acceptable carrier, adjuvant or vehicle. In certain embodiments, these
compositions optionally further comprise one or more additional
therapeutic agents.

[0157] It will also be appreciated that certain of the compounds of
present invention can exist in free form for treatment, or where
appropriate, as a pharmaceutically acceptable derivative or a prodrug
thereof. According to the present invention, a pharmaceutically
acceptable derivative or a prodrug includes, but is not limited to,
pharmaceutically acceptable salts, esters, salts of such esters, or any
other adduct or derivative which upon administration to a patient in need
thereof is capable of providing, directly or indirectly, a compound as
otherwise described herein, or a metabolite or residue thereof.

[0158] As used herein, the term "pharmaceutically acceptable salt" refers
to those salts which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of humans and lower animals
without undue toxicity, irritation, allergic response and the like, and
are commensurate with a reasonable benefit/risk ratio. A
"pharmaceutically acceptable salt" means any non-toxic salt or salt of an
ester of a compound of this invention that, upon administration to a
recipient, is capable of providing, either directly or indirectly, a
compound of this invention or an inhibitorily active metabolite or
residue thereof.

[0159] Pharmaceutically acceptable salts are well known in the art. For
example, S. M. Berge, et al. describes pharmaceutically acceptable salts
in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated
herein by reference. Pharmaceutically acceptable salts of the compounds
of this invention include those derived from suitable inorganic and
organic acids and bases. Examples of pharmaceutically acceptable,
nontoxic acid addition salts are salts of an amino group formed with
inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric
acid, sulfuric acid and perchloric acid or with organic acids such as
acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,
succinic acid or malonic acid or by using other methods used in the art
such as ion exchange.

[0161] As described above, the pharmaceutically acceptable compositions of
the present invention additionally comprise a pharmaceutically acceptable
carrier, adjuvant, or vehicle, which, as used herein, includes any and
all solvents, diluents, or other liquid vehicle, dispersion or suspension
aids, surface active agents, isotonic agents, thickening or emulsifying
agents, preservatives, solid binders, lubricants and the like, as suited
to the particular dosage form desired. Remington's Pharmaceutical
Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton,
Pa., 1980) discloses various carriers used in formulating
pharmaceutically acceptable compositions and known techniques for the
preparation thereof. Except insofar as any conventional carrier medium is
incompatible with the compounds of the invention, such as by producing
any undesirable biological effect or otherwise interacting in a
deleterious manner with any other component(s) of the pharmaceutically
acceptable composition, its use is contemplated to be within the scope of
this invention. Some examples of materials which can serve as
pharmaceutically acceptable carriers include, but are not limited to, ion
exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as
human serum albumin, buffer substances such as phosphates, glycine,
sorbic acid, or potassium sorbate, partial glyceride mixtures of
saturated vegetable fatty acids, water, salts or electrolytes, such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, wool fat, sugars such as
lactose, glucose and sucrose; starches such as corn starch and potato
starch; cellulose and its derivatives such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;
malt; gelatin; talc; excipients such as cocoa butter and suppository
waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame
oil; olive oil; corn oil and soybean oil; glycols; such a propylene
glycol or polyethylene glycol; esters such as ethyl oleate and ethyl
laurate; agar; buffering agents such as magnesium hydroxide and aluminum
hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's
solution; ethyl alcohol, and phosphate buffer solutions, as well as other
non-toxic compatible lubricants such as sodium lauryl sulfate and
magnesium stearate, as well as coloring agents, releasing agents, coating
agents, sweetening, flavoring and perfuming agents, preservatives and
antioxidants can also be present in the composition, according to the
judgment of the formulator.

Uses of Compounds and Pharmaceutically Acceptable Compositions

[0162] In yet another aspect, the present invention provides a method of
treating or lessening the severity of a condition, disease, or disorder
implicated by CFTR mutation. In certain embodiments, the present
invention provides a method of treating a condition, disease, or disorder
implicated by a deficiency of the CFTR activity, the method comprising
administering a composition comprising a compound of Formula (I) to a
subject, preferably a mammal, in need thereof.

[0166] According to an alternative preferred embodiment, the present
invention provides a method of treating cystic fibrosis comprising the
step of administering to said mammal a composition comprising the step of
administering to said mammal an effective amount of a composition
comprising a compound of the present invention.

[0167] According to the invention an "effective amount" of the compound or
pharmaceutically acceptable composition is that amount effective for
treating or lessening the severity of one or more of the diseases,
disorders or conditions as recited above.

[0168] The compounds and compositions, according to the method of the
present invention, may be administered using any amount and any route of
administration effective for treating or lessening the severity of one or
more of the diseases, disorders or conditions as recited above.

[0169] In certain embodiments, the compounds and compositions of the
present invention are useful for treating or lessening the severity of
cystic fibrosis in patients who exhibit residual CFTR activity in the
apical membrane of respiratory and non-respiratory epithelia. The
presence of residual CFTR activity at the epithelial surface can be
readily detected using methods known in the art, e.g., standard
electrophysiological, biochemical, or histochemical techniques. Such
methods identify CFTR activity using in vivo or ex vivo
electrophysiological techniques, measurement of sweat or salivary
Cl.sup.- concentrations, or ex vivo biochemical or histochemical
techniques to monitor cell surface density. Using such methods, residual
CFTR activity can be readily detected in patients heterozygous or
homozygous for a variety of different mutations, including patients
homozygous or heterozygous for the most common mutation, ΔF508.

[0170] In another embodiment, the compounds and compositions of the
present invention are useful for treating or lessening the severity of
cystic fibrosis in patients who have residual CFTR activity induced or
augmented using pharmacological methods or gene therapy. Such methods
increase the amount of CFTR present at the cell surface, thereby inducing
a hitherto absent CFTR activity in a patient or augmenting the existing
level of residual CFTR activity in a patient.

[0171] In one embodiment, the compounds and compositions of the present
invention are useful for treating or lessening the severity of cystic
fibrosis in patients within certain genotypes exhibiting residual CFTR
activity, e.g., class III mutations (impaired regulation or gating),
class IV mutations (altered conductance), or class V mutations (reduced
synthesis) (Lee R. Choo-Kang, Pamela L., Zeitlin, Type I, II, III, IV,
and V cystic fibrosis Tansmembrane Conductance Regulator Defects and
Opportunities of Therapy; Current Opinion in Pulmonary Medicine
6:521-529, 2000). Other patient genotypes that exhibit residual CFTR
activity include patients homozygous for one of these classes or
heterozygous with any other class of mutations, including class I
mutations, class II mutations, or a mutation that lacks classification.

[0172] In one embodiment, the compounds and compositions of the present
invention are useful for treating or lessening the severity of cystic
fibrosis in patients within certain clinical phenotypes, e.g., a moderate
to mild clinical phenotype that typically correlates with the amount of
residual CFTR activity in the apical membrane of epithelia. Such
phenotypes include patients exhibiting pancreatic insufficiency or
patients diagnosed with idiopathic pancreatitis and congenital bilateral
absence of the vas deferens, or mild lung disease.

[0173] The exact amount required will vary from subject to subject,
depending on the species, age, and general condition of the subject, the
severity of the infection, the particular agent, its mode of
administration, and the like. The compounds of the invention are
preferably formulated in dosage unit form for ease of administration and
uniformity of dosage. The expression "dosage unit form" as used herein
refers to a physically discrete unit of agent appropriate for the patient
to be treated. It will be understood, however, that the total daily usage
of the compounds and compositions of the present invention will be
decided by the attending physician within the scope of sound medical
judgment. The specific effective dose level for any particular patient or
organism will depend upon a variety of factors including the disorder
being treated and the severity of the disorder; the activity of the
specific compound employed; the specific composition employed; the age,
body weight, general health, sex and diet of the patient; the time of
administration, route of administration, and rate of excretion of the
specific compound employed; the duration of the treatment; drugs used in
combination or coincidental with the specific compound employed, and like
factors well known in the medical arts. The term "patient", as used
herein, means an animal, preferably a mammal, and most preferably a
human.

[0174] The pharmaceutically acceptable compositions of this invention can
be administered to humans and other animals orally, rectally,
parenterally, intracisternally, intravaginally, intraperitoneally,
topically (as by powders, ointments, drops or patch), bucally, as an oral
or nasal spray, or the like, depending on the severity of the infection
being treated. In certain embodiments, the compounds of the invention may
be administered orally or parenterally at dosage levels of about 0.01
mg/kg to about 50 mg/kg and preferably from about 0.5 mg/kg to about 25
mg/kg, of subject body weight per day, one or more times a day, to obtain
the desired therapeutic effect.

[0176] Injectable preparations, for example, sterile injectable aqueous or
oleaginous suspensions may be formulated according to the known art using
suitable dispersing or wetting agents and suspending agents. The sterile
injectable preparation may also be a sterile injectable solution,
suspension or emulsion in a nontoxic parenterally acceptable diluent or
solvent, for example, as a solution in 1,3-butanediol. Among the
acceptable vehicles and solvents that may be employed are water, Ringer's
solution, U.S.P. and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil can be employed
including synthetic mono- or diglycerides. In addition, fatty acids such
as oleic acid are used in the preparation of injectables.

[0177] The injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions which can be
dissolved or dispersed in sterile water or other sterile injectable
medium prior to use.

[0178] In order to prolong the effect of a compound of the present
invention, it is often desirable to slow the absorption of the compound
from subcutaneous or intramuscular injection. This may be accomplished by
the use of a liquid suspension of crystalline or amorphous material with
poor water solubility. The rate of absorption of the compound then
depends upon its rate of dissolution that, in turn, may depend upon
crystal size and crystalline form. Alternatively, delayed absorption of a
parenterally administered compound form is accomplished by dissolving or
suspending the compound in an oil vehicle. Injectable depot forms are
made by forming microencapsule matrices of the compound in biodegradable
polymers such as polylactide-polyglycolide. Depending upon the ratio of
compound to polymer and the nature of the particular polymer employed,
the rate of compound release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and poly(anhydrides).
Depot injectable formulations are also prepared by entrapping the
compound in liposomes or microemulsions that are compatible with body
tissues.

[0179] Compositions for rectal or vaginal administration are preferably
suppositories which can be prepared by mixing the compounds of this
invention with suitable non-irritating excipients or carriers such as
cocoa butter, polyethylene glycol or a suppository wax which are solid at
ambient temperature but liquid at body temperature and therefore melt in
the rectum or vaginal cavity and release the active compound.

[0180] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms, the
active compound is mixed with at least one inert, pharmaceutically
acceptable excipient or carrier such as sodium citrate or dicalcium
phosphate and/or a) fillers or extenders such as starches, lactose,
sucrose, glucose, mannitol, and silicic acid, b) binders such as, for
example, carboxymethylcellulose, alginates, gelatin,
polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as
glycerol, d) disintegrating agents such as agar-agar, calcium carbonate,
potato or tapioca starch, alginic acid, certain silicates, and sodium
carbonate, e) solution retarding agents such as paraffin, f) absorption
accelerators such as quaternary ammonium compounds, g) wetting agents
such as, for example, cetyl alcohol and glycerol monostearate, h)
absorbents such as kaolin and bentonite clay, and i) lubricants such as
talc, calcium stearate, magnesium stearate, solid polyethylene glycols,
sodium lauryl sulfate, and mixtures thereof. In the case of capsules,
tablets and pills, the dosage form may also comprise buffering agents.

[0181] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such excipients as
lactose or milk sugar as well as high molecular weight polyethylene
glycols and the like. The solid dosage forms of tablets, dragees,
capsules, pills, and granules can be prepared with coatings and shells
such as enteric coatings and other coatings well known in the
pharmaceutical formulating art. They may optionally contain opacifying
agents and can also be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain part of the
intestinal tract, optionally, in a delayed manner. Examples of embedding
compositions that can be used include polymeric substances and waxes.
Solid compositions of a similar type may also be employed as fillers in
soft and hard-filled gelatin capsules using such excipients as lactose or
milk sugar as well as high molecular weight polethylene glycols and the
like.

[0182] The active compounds can also be in microencapsulated form with one
or more excipients as noted above. The solid dosage forms of tablets,
dragees, capsules, pills, and granules can be prepared with coatings and
shells such as enteric coatings, release controlling coatings and other
coatings well known in the pharmaceutical formulating art. In such solid
dosage forms the active compound may be admixed with at least one inert
diluent such as sucrose, lactose or starch. Such dosage forms may also
comprise, as is normal practice, additional substances other than inert
diluents, e.g., tableting lubricants and other tableting aids such a
magnesium stearate and microcrystalline cellulose. In the case of
capsules, tablets and pills, the dosage forms may also comprise buffering
agents. They may optionally contain opacifying agents and can also be of
a composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract, optionally, in
a delayed manner. Examples of embedding compositions that can be used
include polymeric substances and waxes.

[0183] Dosage forms for topical or transdermal administration of a
compound of this invention include ointments, pastes, creams, lotions,
gels, powders, solutions, sprays, inhalants or patches. The active
component is admixed under sterile conditions with a pharmaceutically
acceptable carrier and any needed preservatives or buffers as may be
required. Ophthalmic formulation, eardrops, and eye drops are also
contemplated as being within the scope of this invention. Additionally,
the present invention contemplates the use of transdermal patches, which
have the added advantage of providing controlled delivery of a compound
to the body. Such dosage forms are prepared by dissolving or dispensing
the compound in the proper medium. Absorption enhancers can also be used
to increase the flux of the compound across the skin. The rate can be
controlled by either providing a rate controlling membrane or by
dispersing the compound in a polymer matrix or gel.

[0184] The activity of a compound utilized in this invention as a
modulator of CFTR may be assayed according to methods described generally
in the art and in the Examples herein.

[0185] It will also be appreciated that the compounds and pharmaceutically
acceptable compositions of the present invention can be employed in
combination therapies, that is, the compounds and pharmaceutically
acceptable compositions can be administered concurrently with, prior to,
or subsequent to, one or more other desired therapeutics or medical
procedures. The particular combination of therapies (therapeutics or
procedures) to employ in a combination regimen will take into account
compatibility of the desired therapeutics and/or procedures and the
desired therapeutic effect to be achieved. It will also be appreciated
that the therapies employed may achieve a desired effect for the same
disorder (for example, an inventive compound may be administered
concurrently with another agent used to treat the same disorder), or they
may achieve different effects (e.g., control of any adverse effects). As
used herein, additional therapeutic agents that are normally administered
to treat or prevent a particular disease, or condition, are known as
"appropriate for the disease, or condition, being treated."

[0186] In one embodiment, the additional agent is selected from a
mucolytic agent, a bronchodialator, an anti-biotic, an anti-infective
agent, an anti-inflammatory agent, a CFTR modulator other than a compound
of the present invention, or a nutritional agent. In a further
embodiment, the additional agent is a CFTR modulator other than a
compound of the present invention.

[0187] In one embodiment, the additional agent is an antibiotic. Exemplary
antibiotics useful herein include tobramycin, including tobramycin
inhaled powder (TIP), azithromycin, aztreonam, including the aerosolized
form of aztreonam, amikacin, including liposomal formulations thereof,
ciprofloxacin, including formulations thereof suitable for administration
by inhalation, levoflaxacin, including aerosolized formulations thereof,
and combinations of two antibiotics, e.g., fosfomycin and tobramycin.

[0188] In another embodiment, the additional agent is a mucolyte.
Exemplary mucolytes useful herein includes Pulmozyme®.

[0189] In another embodiment, the additional agent is a bronchodialator.
Exemplary bronchodialtors include albuterol, metaprotenerol sulfate,
pirbuterol acetate, salmeterol, or tetrabuline sulfate.

[0190] In another embodiment, the additional agent is effective in
restoring lung airway surface liquid. Such agents improve the movement of
salt in and out of cells, allowing mucus in the lung airway to be more
hydrated and, therefore, cleared more easily. Exemplary such agents
include hypertonic saline, denufosol tetrasodium
([[(3S,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy-h-
ydroxyphosphoryl][[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydrox-
yoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]hydrogen
phosphate), or bronchitol (inhaled formulation of mannitol).

[0191] In another embodiment, the additional agent is an anti-inflammatory
agent, i.e., an agent that can reduce the inflammation in the lungs.
Exemplary such agents useful herein include ibuprofen, docosahexanoic
acid (DHA), sildenafil, inhaled glutathione, pioglitazone,
hydroxychloroquine, or simavastatin.

[0192] In another embodiment, the additional agent reduces the activity of
the epithelial sodium channel blocker (ENaC) either directly by blocking
the channel or indirectly by modulation of proteases that lead to an
increase in ENaC activity (e.g., seine proteases, channel-activating
proteases). Exemplary such agents include camostat (a trypsin-like
protease inhibitor), QAU145, 552-02, GS-9411, INO-4995, Aerolytic, and
amiloride. Additional agents that reduce the activity of the epithelial
sodium channel blocker (ENaC) can be found, for example in PCT
Publication No. WO2009/074575, the entire contents of which are
incorporated herein in their entirety.

[0194] Combinations of CFTR modulators, such compound of Formula I, and
agents that reduce the activity of ENaC are also useful for treating
diseases mediated by blockade of the epithelial sodium channel also
include diseases other than respiratory diseases that are associated with
abnormal fluid regulation across an epithelium, perhaps involving
abnormal physiology of the protective surface liquids on their surface,
e.g., xerostomia (dry mouth) or keratoconjunctivitis sire (dry eye).
Furthermore, blockade of the epithelial sodium channel in the kidney
could be used to promote diuresis and thereby induce a hypotensive
effect.

[0195] Asthma includes both intrinsic (non-allergic) asthma and extrinsic
(allergic) asthma, mild asthma, moderate asthma, severe asthma,
bronchitic asthma, exercise-induced asthma, occupational asthma and
asthma induced following bacterial infection. Treatment of asthma is also
to be understood as embracing treatment of subjects, e.g., of less than 4
or 5 years of age, exhibiting wheezing symptoms and diagnosed or
diagnosable as "wheezy infants", an established patient category of major
medical concern and now often identified as incipient or early-phase
asthmatics. (For convenience this particular asthmatic condition is
referred to as "wheezy-infant syndrome".) Prophylactic efficacy in the
treatment of asthma will be evidenced by reduced frequency or severity of
symptomatic attack, e.g., of acute asthmatic or bronchoconstrictor
attack, improvement in lung function or improved airways hyperreactivity.
It may further be evidenced by reduced requirement for other, symptomatic
therapy, i.e., therapy for or intended to restrict or abort symptomatic
attack when it occurs, e.g., anti-inflammatory (e.g., cortico-steroid) or
bronchodilatory. Prophylactic benefit in asthma may, in particular, be
apparent in subjects prone to "morning dipping". "Morning dipping" is a
recognized asthmatic syndrome, common to a substantial percentage of
asthmatics and characterized by asthma attack, e.g., between the hours of
about 4-6 am, i.e., at a time normally substantially distant from any
previously administered symptomatic asthma therapy.

[0196] Chronic obstructive pulmonary disease includes chronic bronchitis
or dyspnea associated therewith, emphysema, as well as exacerbation of
airways hyperreactivity consequent to other drug therapy, in particular,
other inhaled drug therapy. In some embodiments, the combinations of CFTR
modulators, such as compounds of Formula I, and agents that reduce the
activity of ENaC are useful for the treatment of bronchitis of whatever
type or genesis including, e.g., acute, arachidic, catarrhal, croupus,
chronic or phthinoid bronchitis.

[0197] In another embodiment, the additional agent is a CFTR modulator
other than a compound of formula I, i.e., an agent that has the effect of
modulating CFTR activity. Exemplary such agents include ataluren
("PTC124®"; 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid),
sinapultide, lancovutide, depelestat (a human recombinant neutrophil
elastase inhibitor), cobiprostone
(7-{(2R,4aR,5R,7aR)-2-[(3S)-1,1-difluoro-3-methylpentyl]-2-hydroxy-6-oxoo-
ctahydrocyclopenta[b]pyran-5-yl}heptanoic acid), or
(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-
-methylpyridin-2-yl)benzoic acid. In another embodiment, the additional
agent is (3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbo-
xamido)-3-methylpyridin-2-yl)benzoic acid.

[0198] In another embodiment, the additional agent is a nutritional agent.
Exemplary such agents include pancrelipase (pancreating enzyme
replacement), including Pancrease®, Pancreacarb®, Ultrase®,
or Creon®, Liprotomase® (formerly Trizytek®), Aquadeks®,
or glutathione inhalation. In one embodiment, the additional nutritional
agent is pancrelipase.

[0199] The amount of additional therapeutic agent present in the
compositions of this invention will be no more than the amount that would
normally be administered in a composition comprising that therapeutic
agent as the only active agent. Preferably the amount of additional
therapeutic agent in the presently disclosed compositions will range from
about 50% to 100% of the amount normally present in a composition
comprising that agent as the only therapeutically active agent.

[0200] The compounds of this invention or pharmaceutically acceptable
compositions thereof may also be incorporated into compositions for
coating an implantable medical device, such as prostheses, artificial
valves, vascular grafts, stents and catheters. Accordingly, the present
invention, in another aspect, includes a composition for coating an
implantable device comprising a compound of the present invention as
described generally above, and in classes and subclasses herein, and a
carrier suitable for coating said implantable device. In still another
aspect, the present invention includes an implantable device coated with
a composition comprising a compound of the present invention as described
generally above, and in classes and subclasses herein, and a carrier
suitable for coating said implantable device. Suitable coatings and the
general preparation of coated implantable devices are described in U.S.
Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings are typically
biocompatible polymeric materials such as a hydrogel polymer,
polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic
acid, ethylene vinyl acetate, and mixtures thereof. The coatings may
optionally be further covered by a suitable topcoat of fluorosilicone,
polysaccarides, polyethylene glycol, phospholipids or combinations
thereof to impart controlled release characteristics in the composition.

[0201] Another aspect of the invention relates to modulating CFTR activity
in a biological sample or a patient (e.g., in vitro or in vivo), which
method comprises administering to the patient, or contacting said
biological sample with a compound of Formula (I) or a composition
comprising said compound. The term "biological sample", as used herein,
includes, without limitation, cell cultures or extracts thereof; biopsied
material obtained from a mammal or extracts thereof; and blood, saliva,
urine, feces, semen, tears, or other body fluids or extracts thereof.

[0202] Modulation of CFTR in a biological sample is useful for a variety
of purposes that are known to one of skill in the art. Examples of such
purposes include, but are not limited to, the study of CFTR in biological
and pathological phenomena; and the comparative evaluation of new
modulators of CFTR.

[0203] In yet another embodiment, a method of modulating activity of an
anion channel in vitro or in vivo, is provided comprising the step of
contacting said channel with a compound of Formula (I). In preferred
embodiments, the anion channel is a chloride channel or a bicarbonate
channel. In other preferred embodiments, the anion channel is a chloride
channel.

[0204] According to an alternative embodiment, the present invention
provides a method of increasing the number of functional CFTR in a
membrane of a cell, comprising the step of contacting said cell with a
compound of Formula (I).

[0205] According to another preferred embodiment, the activity of the CFTR
is measured by measuring the transmembrane voltage potential. Means for
measuring the voltage potential across a membrane in the biological
sample may employ any of the known methods in the art, such as optical
membrane potential assay or other electrophysiological methods.

[0207] These voltage sensitive assays are based on the change in
fluorescence resonant energy transfer (FRET) between the
membrane-soluble, voltage-sensitive dye, DiSBAC2(3), and a
fluorescent phospholipid, CC2-DMPE, which is attached to the outer
leaflet of the plasma membrane and acts as a FRET donor. Changes in
membrane potential (Vm) cause the negatively charged DiSBAC2(3)
to redistribute across the plasma membrane and the amount of energy
transfer from CC2-DMPE changes accordingly. The changes in fluorescence
emission can be monitored using VIPR® II, which is an integrated
liquid handler and fluorescent detector designed to conduct cell-based
screens in 96- or 384-well microtiter plates.

[0208] In another aspect the present invention provides a kit for use in
measuring the activity of CFTR or a fragment thereof in a biological
sample in vitro or in vivo comprising (i) a composition comprising a
compound of Formula (I) or any of the above embodiments; and (ii)
instructions for a) contacting the composition with the biological sample
and b) measuring activity of said CFTR or a fragment thereof. In one
embodiment, the kit further comprises instructions for a) contacting an
additional composition with the biological sample; b) measuring the
activity of said CFTR or a fragment thereof in the presence of said
additional compound, and c) comparing the activity of the CFTR in the
presence of the additional compound with the density of the CFTR in the
presence of a composition of Formula (I). In preferred embodiments, the
kit is used to measure the density of CFTR.

[0209] In order that the invention described herein may be more fully
understood, the following examples are set forth. It should be understood
that these examples are for illustrative purposes only and are not to be
construed as limiting this invention in any manner.

Processes and Intermediates for Making Compounds of Formula I

[0210] Another aspect of the invention relates to a process for preparing
a compound of Formula (Ic):

##STR00051## [0211] or pharmaceutically acceptable salts thereof,
wherein the process comprises: [0212] (a) reacting the acid of formula 1d
with an amine of formula 2d to provide a compound of formula (Ic)

[0212] ##STR00052## [0213] wherein: [0214] ring A is selected from:

[0214] ##STR00053## [0215] wherein: [0216] R1 is --CH3,
--CF3 or --CN; [0217] R2 is hydrogen, --CH3, --CF3,
--OH, or --CH2OH; [0218] R3 is hydrogen, --CH3,
--OCH3, or --CN; [0219] provided that both R2 and R3 are
not simultaneously hydrogen, and [0220] Ra is hydrogen or a silyl
protecting group selected from the group consisting of trimethylsilyl
(TMS), tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBDMS)
triisopropylsilyl (TIPS), and [2-(trimethylsilyl)ethoxy]methyl (SEM); and
[0221] one of X and Y is nitrogen and the other is carbon.

[0222] In one embodiment, the reaction of the acid of formula 1d with the
amine of formula 2c occurs in a solvent in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and triethylamine or in a solvent in the
presence of propyl phosphonic acid cyclic anhydride (T3P®) and
pyridine. More particularly, the solvent comprises N,N-dimethyl
formamide, ethyl acetate or 2-methyltetrahydrofuran.

[0223] In another embodiment, Ra is hydrogen or TBDMS.

[0224] In another embodiment, Ra is TBDMS.

[0225] In another embodiment, the process comprises a further deprotection
step; for instance, when ring A is

##STR00054##

wherein Ra is a silyl protecting group, to generate a compound of
formula I, wherein ring A is

##STR00055##

Typically, removal of a silyl protecting group requires treatment with
acid such as acetic acid or a dilute mineral acid or the like, although
other reagents, such as a source of fluoride ion (e.g.,
tetrabutylammonium fluoride), may be used.

[0226] In the process, the amine of formula 2c is prepared from a compound
of formula 2a comprising the steps of: [0227] (a) reacting the compound
of formula 2a with an amine of formula 3 to provide the compound of
formula 2b

[0227] ##STR00056## [0228] wherein: [0229] Hal is F, Cl, Br, or I;
and [0230] the amine of formula 3 is

##STR00057##

[0230] and [0231] (b) reducing the compound of formula 2b to the amine
of formula 2c.

##STR00058##

[0232] In one embodiment of the process for making the amine of formula
2c, the amine of formula 3 in step (a) is generated in situ from the
corresponding quaternary ammonium salt, such as an amine hydrochloride
salt, although other ammonium salts (e.g. the trifluoracetate salt), may
be used as well.

[0233] In one embodiment of step (a) for forming the amine of formula 2c,
when the amine of formula 3 is

##STR00059##

Ra is hydrogen or TBDMS. More particularly, Ra is TBDMS.

[0234] In another embodiment, step (a) occurs in a polar aprotic solvent
in the presence of a tertiary amine base. Examples of solvents that can
be employed include N,N-dimethyl formamide, dimethyl sulfoxide or
acetonitrile. Examples of tertiary amines that can be employed include
triethylamine, diisopropylethyl amine, 1,5-diazabicyclo[4.3.0]non-5-ene
(DBN), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),
1,4-diazabicyclo[2.2.2]octane (DABCO) and pyridine.

[0235] In one embodiment, the tertiary amine base is triethylamine.

[0236] In another embodiment, step (a) occurs in acetonitrile in the
presence of triethylamine.

[0237] In another embodiment, the reaction temperature of step (a) is
between approximately 75° C. and approximately 85° C.

[0238] In another embodiment, the reaction time for step (a) is between
approximately 2 and approximately 30 hours.

[0239] In one embodiment of the process for making the amine of formula
2c, step (b) occurs in a polar protic solvent or a mixture of polar
protic solvents in the presence of a palladium catalyst. When palladium
is the catalyst, the solvent in step (b) typically is a polar protic
solvent such as an alcohol. More particularly, the solvent comprises
methanol or ethanol.

[0240] In another embodiment, step (b) occurs in a polar protic solvent,
such as water, in the presence of Fe and FeSO4 or Zn and AcOH.

[0241] Another aspect of the invention relates to a process for preparing
a compound of Formula Ic:

##STR00060## [0242] or pharmaceutically acceptable salts thereof,
comprising the steps of: [0243] (a) reacting a compound of formula 2a
with an amine of formula 3 to provide a compound of formula 2b

[0248] ##STR00065## [0249] wherein: [0250] R1 is --CH3,
--CF3 or --CN; [0251] R2 is hydrogen, --CH3, --CF3,
--OH, or --CH2OH; [0252] R3 is hydrogen, --CH3,
--OCH3, or --CN; [0253] provided that both R2 and R3 are
not simultaneously hydrogen; [0254] Ra is hydrogen or a silyl
protecting group selected from the group consisting of trimethylsilyl
(TMS), tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBDMS),
triisopropylsilyl (TIPS), and [2-(trimethylsilyl)ethoxy]methyl (SEM); and
[0255] one of X and Y is nitrogen and the other is carbon.

[0256] In one embodiment, the amine of formula 3 in step (a) is generated
in situ from the corresponding quaternary ammonium salt, such as an amine
hydrochloride salt, although other ammonium salts (e.g. the
trifluoracetate salt), may be used as well.

[0257] In one embodiment of step (a) for forming the amine of formula 2c,
when the amine of formula 3 is

##STR00066##

Ra is hydrogen or TBDMS. More particularly, Ra is TBDMS.

[0258] In another embodiment, step (a) occurs in a polar aprotic solvent
in the presence of a tertiary amine base. Examples of solvents that can
be employed include N,N-dimethyl formamide, dimethyl sulfoxide or
acetonitrile. Examples of tertiary amines that can be employed include
triethylamine, diisopropylethyl amine, 1,5-diazabicyclo[4.3.0]non-5-ene
(DBN), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),
1,4-diazabicyclo[2.2.2]octane (DABCO) and pyridine.

[0259] In one embodiment, the tertiary amine base is triethylamine.

[0260] In another embodiment, step (a) occurs in acetonitrile in the
presence of triethylamine.

[0261] In another embodiment, the reaction temperature of step (a) is
between approximately 75° C. and approximately 85° C.

[0262] In another embodiment, the reaction time for step (a) is between
approximately 2 and approximately 30 hours.

[0263] In one embodiment of the process for making the amine of formula
2c, step (b) occurs in a polar protic solvent or a mixture of polar
protic solvents in the presence of a palladium catalyst. When palladium
is the catalyst, the solvent in step (b) typically is a polar protic
solvent such as an alcohol. More particularly, comprises methanol or
ethanol.

[0264] In another embodiment, step (b) occurs in a polar protic solvent,
such as water, in the presence of Fe and FeSO4 or Zn and AcOH.

[0265] In one embodiment of step (c), the reaction of the acid of formula
1d with the amine of formula 2c occurs in a solvent in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and triethylamine or in a solvent in the
presence of propyl phosphonic acid cyclic anhydride (T3P®) and
pyridine. More particularly, the solvent comprises N,N-dimethyl
formamide, ethyl acetate, or 2-methyltetrahydrofuran.

[0266] In another embodiment, Ra is hydrogen or TBDMS.

[0267] In another embodiment, Ra is TBDMS.

[0268] In another embodiment, the process comprises a further deprotection
step; for instance, when ring A is

##STR00067##

wherein Ra is a silyl protecting group, to generate a compound of
formula I, wherein ring A is

##STR00068##

Typically, removal of a silyl protecting group requires treatment with
acid such as acetic acid or a dilute mineral acid or the like, although
other reagents, such as a source of fluoride ion (e.g.,
tetrabutylammonium fluoride), may be used.

[0269] Another aspect of the invention relates to a compound which is

##STR00069##

wherein ring A is

##STR00070##

wherein [0270] R1 is --CH3, --CF3 or --CN, [0271]
Ra is hydrogen or a silyl protecting group selected from the group
consisting of trimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS),
tert-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), and
[2-(trimethylsilyl)ethoxy]methyl (SEM); and one of X and Y is nitrogen
and the other is carbon.

[0272] Another aspect of the invention relates to a compound which is

##STR00071##

wherein ring A is

##STR00072##

wherein [0273] R1 is --CH3, --CF3 or --CN, [0274]
Ra is hydrogen or a silyl protecting group selected from the group
consisting of trimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS),
tert-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), and
[2-(trimethylsilyl)ethoxy]methyl (SEM); and one of X and Y is nitrogen
and the other is carbon.

[0275] Another aspect of the invention relates to a compound of formula
Ic:

[0277] ##STR00074## wherein [0278] R1 is --CH3,
--CF3 or --CN; [0279] R2 is hydrogen, --CH3, --CF3,
--OH, or --CH2OH; [0280] R3 is hydrogen, --CH3,
--OCH3, or --CN; [0281] provided that both R2 and R3 are
not simultaneously hydrogen, and [0282] Ra is a silyl protecting
group selected from the group consisting of trimethylsilyl (TMS),
tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBDMS) and
triisopropylsilyl (TIPS), and [2-(trimethylsilyl)ethoxy]methyl (SEM); and
[0283] one of X and Y is nitrogen and the other is carbon.

[0284] Another aspect of the invention relates to a compound of formula I

[0293] Another aspect of the invention relates to a compound selected from
the group consisting of:

##STR00077##

made by any of the processes disclosed herein.

General Synthetic Schemes

[0294] Schemes 1-3 illustrate the synthesis of compounds of Formula (I) of
the present invention.

##STR00078##

[0295] Scheme 1 depicts a convergent approach to the preparation of
compounds of Formula (I) from 1a and 2a. In the ultimate transformation,
amide formation via coupling of carboxylic acid 1d with amine 2c to give
a compound of formula (I) can be achieved using either
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and triethylamine in N,N-dimethyl formamide
(DMF) or propyl sulfronic acid cyclic anhydride (T3P®) and pyridine
in 2-methyltetrahydrofuran. Carboxylic acid 1d is prepared from the
corresponding substituted benzene derivative 1a via a sequence commencing
with heat-mediated condensation of 1a with an appropriate malonate
(CO2R)2CH═CH(OR), wherein R is an alkyl group such as
methyl, ethyl, or the like, to provide 1b.

[0296] Compound 1b is converted to carboxylic acid 1d via a three step
sequence including intramolecular cyclization upon heating at reflux in
Dowtherm or diphenyl ether (step b), followed by removal (if needed) of
the blocking halo group (step c) under palladium-catalyzed dehalogenation
conditions and acid- or base-catalyzed saponification (step d). The order
of the deprotection and saponification steps can be reversed; i.e., step
c can occur before or after step d, as depicted in Scheme 1.

[0297] Referring again to Scheme 1, aniline derivative 2c can be prepared
from nitro compound 2a via a three step sequence. Thus, coupling of 2a
with a cyclic amine

[0298] Scheme 2 depicts the synthesis of a compounds of formula (I)
bearing a propynyl amine sidechain. Thus, coupling of nitrobenzene 2a,
wherein Hal is bromide, chloride, or the like, with

##STR00081##

3 as defined herein in the presence potassium carbonate in DMSO provides
compound 4. Palladium-catalyzed coupling of compound 4 with
N,N-dimethylprop-2-yn-1-amine, followed by iron or zinc catalyzed
reduction of the nitro moiety, provides amine 5. Coupling of amine 5 with
carboxylic acid 1d provides compound 6 which is a compound of formula
(I).

##STR00082##

[0299] Scheme 3 depicts the synthesis of a compound of formula (I) wherein

[0300] Thus, as with the series of transformations summarized in Schemes 1
and 2, coupling of compound 2a with the bicyclo[2.2.1]amine 7 provides
compound 8. If a hydroxy group is present in compound 8, it may be
necessary to protect the hydroxy group with a protecting group prior to
subsequent transformations. Thus, treatment of compound 8 with tert-butyl
dimethylsilyl chloride using known conditions provides the protected
compound 9 prior to reduction of the nitro moiety to provide the amine
10. Amide formation with 1d (cf. Scheme 4) and removal of the hydroxy
protecting group (as needed) provides compound 11 which is a compound of
Formula (I).

[0304] A 3-neck, 5-L flask was charged with of ethyl
8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylate 15
(100 g, 0.3 mol), ethanol (1250 mL, 12.5 mL/g) and triethylamine (220 mL,
1.6 mol). The vessel was then charged with 10 g of 10% Pd/C (50% wet) at
5° C. The reaction was stirred vigorously under hydrogen
atmosphere for 20 h at 5° C., after which time the reaction
mixture was concentrated to a volume of approximately 150 mL. The
product, ethyl 4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxylate 16,
as a slurry with Pd/C, was taken directly into the next step.

[0323] The assay utilizes fluorescent voltage sensing dyes to measure
changes in membrane potential using a fluorescent plate reader (e.g.,
FLIPR III, Molecular Devices, Inc.) as a readout for increase in
functional ΔF508-CFTR in NIH 3T3 cells. The driving force for the
response is the creation of a chloride ion gradient in conjunction with
channel activation by a single liquid addition step after the cells have
previously been treated with compounds and subsequently loaded with a
voltage sensing dye.

[0324] Identification of Potentiator Compounds

[0325] To identify potentiators of ΔF508-CFTR, a double-addition HTS
assay format was developed. This HTS assay utilizes fluorescent voltage
sensing dyes to measure changes in membrane potential on the FLIPR III as
a measurement for increase in gating (conductance) of ΔF508 CFTR in
temperature-corrected ΔF508 CFTR NIH 3T3 cells. The driving force
for the response is a CF ion gradient in conjunction with channel
activation with forskolin in a single liquid addition step using a
fluorescent plate reader such as FLIPR III after the cells have
previously been treated with potentiator compounds (or DMSO vehicle
control) and subsequently loaded with a redistribution dye.

[0329] NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are
used for optical measurements of membrane potential. The cells are
maintained at 37° C. in 5% CO2 and 90% humidity in Dulbecco's
modified Eagle's medium supplemented with 2 mM glutamine, 10% fetal
bovine serum, 1×NEAA, β-ME, 1× pen/strep, and 25 mM
HEPES in 175 cm2 culture flasks. For all optical assays, the cells
were seeded at 20,000/well in 384-well matrigel-coated plates and
cultured for 2 hrs at 37° C. before culturing at 27° C. for
24 hrs. for the potentiator assay. For the correction assays, the cells
are cultured at 27° C. or 37° C. with and without compounds
for 16-24 hours. Electrophysiological Assays for assaying
ΔF508-CFTR modulation properties of compounds.

[0330] 1. Using Chamber Assay

[0331] Using chamber experiments were performed on polarized airway
epithelial cells expressing ΔF508-CFTR to further characterize the
ΔF508-CFTR modulators identified in the optical assays. Non-CF and
CF airway epithelia were isolated from bronchial tissue, cultured as
previously described (Galietta, L. J. V., Lantero, S., Gazzolo, A.,
Sacco, O., Romano, L., Rossi, G. A., & Zegarra-Moran, O. (1998) In Vitro
Cell. Dev. Biol. 34, 478-481), and plated onto Costar® Snapwell®
filters that were precoated with NIH3T3-conditioned media. After four
days the apical media was removed and the cells were grown at an air
liquid interface for >14 days prior to use. This resulted in a
monolayer of fully differentiated columnar cells that were ciliated,
features that are characteristic of airway epithelia. Non-CF HBE were
isolated from non-smokers that did not have any known lung disease.
CF-HBE were isolated from patients homozygous for ΔF508-CFTR.

[0334] Typical protocol utilized a basolateral to apical membrane Clconcentration gradient. To set up this gradient, normal ringers was used
on the basolateral membrane, whereas apical NaCl was replaced by
equimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give a large
Cl.sup.- concentration gradient across the epithelium. Forskolin (10
μM) and all test compounds were added to the apical side of the cell
culture inserts. The efficacy of the putative ΔF508-CFTR
potentiators was compared to that of the known potentiator, genistein.

[0338] The ability of ΔF508-CFTR potentiators to increase the
macroscopic ΔF508-CFTR Cl.sup.- current (I.sub.ΔF508) in
NIH3T3 cells stably expressing ΔF508-CFTR was also investigated
using perforated-patch-recording techniques. The potentiators identified
from the optical assays evoked a dose-dependent increase in
IΔ.sub.F508 with similar potency and efficacy observed in the
optical assays. In all cells examined, the reversal potential before and
during potentiator application was around -30 mV, which is the calculated
ECl (-28 mV).

[0339] Cell Culture

[0340] NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are
used for whole-cell recordings. The cells are maintained at 37° C.
in 5% CO2 and 90% humidity in Dulbecco's modified Eagle's medium
supplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA,
β-ME, 1× pen/strep, and 25 mM HEPES in 175 cm2 culture
flasks. For whole-cell recordings, 2,500-5,000 cells were seeded on
poly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at
27° C. before use to test the activity of potentiators; and
incubated with or without the correction compound at 37° C. for
measuring the activity of correctors.

[0341] 3. Single-Channel Recordings

[0342] Gating activity of wt-CFTR and temperature-corrected
ΔF508-CFTR expressed in NIH3T3 cells was observed using excised
inside-out membrane patch recordings as previously described (Dalemans,
W., Barbry, P., Champigny, G., Jallat, S., Dott, K., Dreyer, D., Crystal,
R. G., Pavirani, A., Lecocq, J-P., Lazdunski, M. (1991) Nature 354,
526-528) using an Axopatch 200B patch-clamp amplifier (Axon Instruments
Inc.). The pipette contained (in mM): 150 NMDG, 150 aspartic acid, 5
CaCl2, 2 MgCl2, and 10 HEPES (pH adjusted to 7.35 with Tris
base). The bath contained (in mM): 150 NMDG-C1, 2 MgCl2, 5 EGTA, 10
TES, and 14 Tris base (pH adjusted to 7.35 with HCl). After excision,
both wt- and ΔF508-CFTR were activated by adding 1 mM Mg-ATP, 75 nM
of the catalytic subunit of cAMP-dependent protein kinase (PKA; Promega
Corp. Madison, Wis.), and 10 mM NaF to inhibit protein phosphatases,
which prevented current rundown. The pipette potential was maintained at
80 mV. Channel activity was analyzed from membrane patches containing 2
active channels. The maximum number of simultaneous openings determined
the number of active channels during the course of an experiment. To
determine the single-channel current amplitude, the data recorded from
120 sec of ΔF508-CFTR activity was filtered "off-line" at 100 Hz
and then used to construct all-point amplitude histograms that were
fitted with multigaussian functions using Bio-Patch Analysis software
(Bio-Logic Comp. France). The total microscopic current and open
probability (Po) were determined from 120 sec of channel activity.
The Po was determined using the Bio-Patch software or from the
relationship Po=I/i(N), where I=mean current, i=single-channel
current amplitude, and N=number of active channels in patch.

[0345] Compounds of the invention are useful as modulators of ATP binding
cassette transporters. Examples of activities and efficacies of the
compounds of Formula (I) are shown below in Table 3. The compound
activity is illustrated with "+++" if activity was measured to be less
than 2.0 μM, "++" if activity was measured to be from 2 μM to 5.0
μM, "+" if activity was measured to be greater than 5.0 μM, and "-"
if no data was available. The efficacy is illustrated with "+++" if
efficacy was calculated to be greater than 100%, "++" if efficacy was
calculated to be from 100% to 25%, "+" if efficacy was calculated to be
less than 25%, and "-" if no data was available. It should be noted that
100% efficacy is the maximum response obtained with
4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)phenol.